1. Field of the Invention
The present invention relates to a lipid compound modified by a functional group which can be suitably bonded to bioactive substances such as an antigen, an antibody, and an enzyme.
2. Description of the Related Art
The present invention further relates to a lipid composition, containing at least the above lipid compound, and suitable for immobilization of bioactive substances.
The present invention still further relates to a method for immobilizing bioactive substances to the above lipid composition and the lipid composition having the bioactive substances immobilized thereto by this method.
The present invention still further relates to a liposome reagent obtained by immobilizing an antigen or antibody on the surface of a liposome prepared from the above lipid composition. This liposome reagent is effective in immunoassay.
Immunoassay is an analysis method of identifying and quantifying a specific antigen or antibody contained in a sample by utilizing high specificity and sensitivity of an antigen-antibody reaction.
Known examples of a quantitative analysis method according to immunoassay are radioimmunoassay (to be referred to as RIA hereinafter) and immunoelectrophoresis. However, in RIA, since a radioactive element is used, special equipment for dealing with the radioactive element must be used, and an operator having a license for this purpose must operate the equipment. In addition, it is difficult to handle RIA waste material because a radioactive element is used.
Meanwhile, since the sensitivity is low in immunoelectrophoresis, immunoelectrophoresis cannot be applied when the concentration of a target substance is significantly low. In addition, a long time period is required for measurement.
Recently, immunoassay has been performed using a lipid membrane such as a liposome or an LB membrane on the surface of which an antigen or antibody is immobilized, and then introducing a complement. In this immunoassay, the complement is activated by an antigenantibody reaction between the immobilized antigen or antibody and a target substance in a sample, and lysis of the lipid membrane is finally resulted by a cascade reaction of the activated complement. As a reagent for performing immunoassay utilizing such a reaction of a complement, the present inventors have disclosed, in, e.g., Japanese Patent Disclosure (Kokai) No. 60-117159, a liposome reagent on the surface of which a hydrophilic antigen or antibody is immobilized and in which a hydrophilic marker substance is enclosed. This liposome reagent is destroyed by the above reaction in a sample in which an antigen or antibody is present, and the marker substance enclosed therein is released. The amount of the released marker substance can be correlated with that of the target substance. Therefore, the target substance can be quantified by quantifying the released marker substance by a predetermined analysis method (e.g., fluorescent analysis).
In another conventional method, an LB membrane on the surface of which an antigen or antibody is immobilized is used, and a target substance in a sample solution is quantified by an antigen-antibody reaction and a reaction of a complement in the same manner as when the liposome reagent is used.
Furthermore, analysis other than immunoassay can be performed by immobilizing a bioactive substance other than an antigen or antibody, e.g., an enzyme to a lipid membrane such as a liposome or lipid bilayer (LB) membrane.
Conventional methods of immobilizing the bioactive substances such as an enzyme and an antibody on the surface of a lipid membrane are as follows:
a method of using phosphatidyl ethanolamine (DTP-DPPE) modified by N-hydroxysuccinimidyl-3-(2-pyridyldithio)propionate (SPDP) (e.g., L.D. Leserman, J. Barnet, F. Kourilski and J.N. Weinstein; Nature, 288, PP. 602-604 (1980), and F.J. Martin, W.L. Hubbell and D. Papahadjopoulos; Biochemistry, 20, PP. 4229-4238 (1981));
a method of using a lipid to which a thiomaleimide group is introduced (H. Hashimoto, M. Sugawara and H. Endoh; J. Immunol. Methods, 62, PP. 155-162 (1983)); and
a method of using a lipid to which a disulfide group is introduced (Japanese Patent Publication No. 61-45775).
However, the above immobilizing methods have the following problems. That is, in these methods, when immunoassay is performed using a lipid membrane to which an antigen or antibody is immobilized, the lipid membrane is sometimes destroyed when it is mixed with blood or serum regardless of whether a target substance is present. For example, the liposome reagent (disclosed in Japanese Patent Disclosure (Kokai) No. 60-117159) having an antibody immobilized on the surface thereof is significantly destroyed when it is just mixed with blood or serum as a sample, and release of an enclosed substance is found. For this reason, it is impossible to accurately quantify the target substance in the blood because background noise is high. Such a problem is assumed to be caused since a nonspecific reaction occurs between another protein or minor chemical component in the blood or serum and the liposome in addition to the antigen-antibody reaction between the target substance and the liposome.
In order to eliminate such adverse effects of the nonspecific reaction, a serum or protein-containing sample is diluted by, e.g., about 100 times. In this method, however, a target substance which is originally contained at only a low concentration is further diluted before analysis. Therefore, it is sometimes very difficult to precisely quantify the target substance of certain types.